Nutritional and therapeutic composition of an insulin sensitizer and a peptide fraction

ABSTRACT

The present invention discloses a composition comprising an insulin sensitizer and a peptide fraction, which promotes significant reduction in blood glucose levels and stabilizes blood glucose levels in individuals with type 2 diabetes.

FIELD OF THE INVENTION

The present invention relates to a composition comprising an insulinsensitizer.

BACKGROUND OF THE INVENTION

Type 2 diabetes mellitus is a clinically and genetically heterogeneousgroup of syndromes characterized by elevated blood glucose levels. Itoccurs because the insulin produced by the β (beta) cells of thepancreas is either insufficient or ineffectively utilized by targettissues, resulting in high levels of glucose in the blood. Post-prandialpeaks (a rise of glucose in the body after a meal), which usually resultfrom a high carbohydrate diet, contribute to the high blood glucoselevels. Complications that surface from diabetes can usually be tracedback to excessive sugar levels in the blood over a period of many years.

The progression of type 2 diabetes is often characterised as follows. Atfirst a slow but progressive increase in insulin resistance develops.This implies that insulin production by the pancreas is normal duringthe early stages of the disease, but the ability of insulin to increaseglucose uptake is reduced. The reduced insulin sensitivity in the earlystages of the disease is usually compensated by an increased release ofinsulin by the pancreas. However, after several years the pancreas nolonger responds properly to glucose ingestion and the reduced insulinsensitivity is accompanied by a suboptimal insulin production. Theresulting hyperglycaemia (high blood glucose level) leads to a rapiddisabilitating progression of the disease and the necessity to startusing oral medication and finally exogenous insulin therapy.

The abnormally high levels of glucose in the blood may contribute tovarious micro- and macrovascular complications, including cardiovasculardisease, retinopathy, nephropathy and neuropathy. Serious healthcomplications resulting from these high glucose levels include eye,heart, kidney, and nerve damage.

The key to fighting diabetes is through monitoring and controlling bloodsugar levels. If patients conscientiously monitor and control theirblood sugar level at an early stage, they may delay or prevent manycomplications associated with the disorder. A proper diet and exercisecan help people with diabetes to maintain healthy blood glucose levels.However, when diet and exercise are inadequate to control diabetes,medication is required. At this stage treatment can still rely on theuse of oral anti-diabetic drugs alone, i.e. exogenous insulin therapy isnot yet necessary.

In principle three classes of oral anti-diabetes drugs are available,namely the “blocker” class, the “stimulator” class and the “sensitizer”class. The “blocker” class of oral anti-diabetes agents has been shownto delay or prevent further development of the disease. Examples of thisclass of agents are the so-called alpha-glucosidase inhibitors, whichact by delaying the absorption of glucose from the ingestedcarbohydrates. The “stimulator” class of oral anti-diabetic agentsstimulate the production of insulin by the pancreas. Examples of the“stimulator” class are sulfonylureas, which are known to be effective tostimulate secretion of insulin. The “sensitizer” class of oralanti-diabetic agents help to use glucose more efficiently or to maketissue cells more sensitive to insulin. Examples of pharmaceutical“insulin sensitizers” are biguanides (such as Metformin (e.g.Glucophage®) and thiazolidinediones (such as Pioglitazone (e.g. Actos®)and Rosiglitazone (e.g. Avandia®). Not surprisingly several of theseoral anti-diabetic drugs have undesirable side effects. For example,adverse effects of sulphonylurea antidiabetic agents includehypoglycaemia, gastro-intestinal disturbances and hypersensitivityreactions. Adverse effects of the insulin sensitiser biguanide includegastrointestinal disturbances and lactic acidosis.

Milder insulin sensitizing effects can be obtained with a number ofnatural compounds. The obvious advantage of such natural insulinsensitizers is that they are convenient and safe to use. These naturalinsulin sensitizers can, for example, be added to our regular diet, orbe added to dietary supplements and functional foods. One of thesenatural insulin sensitizers is chromium. Chromium is a trace mineralthat is essential for normal insulin function. Dietary studies indicatethat most people in the U.S. and other industrialized countries do notconsume enough chromium, and deficiencies appear to be even more commonin diabetic people. Many clinical studies support the benefits andsafety of chromium supplementation in diabetic people. Supplementalchromium is known to lower blood insulin levels, improve glucosetolerance and decrease haemoglobin glycosylation in people with type 2diabetes. Chromium also helps maintain healthy blood lipid levels, inparticular triglycerides and HDL cholesterol. Experts, such as RichardAnderson, Ph.D. from the U.S. Dept. of Agriculture at the BeltsvilleHuman Nutrition Research Center, recommend chromium supplementation indaily amounts of 200-1000 micrograms. Clinical studies show that inparticular the organic or chelated forms, such as chromiumpolynicotinate, picolinate, glycine-niacin chelate, GTF chromium andchromium yeast are effective. Furthermore it has been shown thatcombinations of chromium and biotin (i.e. Vitamin B8) actsynergistically.

Vanadium is another trace element involved in promoting normal insulinfunction. Some studies have shown that vanadium supplied as vanadylsulfate can improve glucose tolerance in type 2 diabetics.Supplementation with up to 100 micrograms/day is safe, and will satisfythe body's nutritional vanadium needs including the requirement forinsulin and glucose metabolism.

Niacin is another B-vitamin of special importance to diabetes. 100milligrams/day of niacin has been shown to improve glucose tolerance andfasting blood glucose in diabetics when co-supplemented with chromium.

Fenugreek (Trigonella foenum-graecum) is a herb that is native to SouthEast Europe, North Africa and Western Asia. The herb has been shown tobe beneficial in the treatment of both type 1 and type 2 diabetes, andis thought to help control blood sugar in addition to lowering serum.cholesterol levels. A dose as small as 15 mg may help to control fastingblood sugar levels as well as control sudden peaks in blood sugarfollowing meals [‘Gales Encyclopedia of Alternative Medicine’, TumerJ.].

Banaba Leaf Extract is a newcomer among standardized diabetes herbs andis currently sold under the trade name “Glucosol.” The active compoundin Glucosol, corosolic acid, has been shown to promote the transport ofblood glucose into cells. Glucosol is standardized to provide a minimumof 1% corosolic acid. The extract was found to be well-tolerated andsafe in human and animal studies. Compared to other herbal extracts usedbe people with diabetes, Glucosol offers the advantage that it can lowerblood glucose in diabetic people without causing hypoglycaemia. Also,the clinically effective dose is only 32-48 milligrams daily. A seriesof recent and still unpublished clinical studies conducted in 1999 byDr. W. Judy at the Southeastern Institute of Biomedical Research(Bradenton, Fla.), showed significant benefits of Glucosol when takendaily for 30 days at 32 and 48 milligrams per day. One of these studieswas a randomized, double-blind crossover study with 12 diabetic subjectstaking 48milligrams of the extract. Glucosol lowered fasting bloodglucose in people with type 2 diabetes, and the effect was sustained forseveral weeks even after discontinuation of the supplement. Studyreports are available from the manufacturer (Soft Gel Technologies,Inc.) at their website (www.glucosol.com/glucosol/default. htm).

Pterostilbene, a compound found in grapes, has been identified by theUSDA, and is a phytoalexin that is produced by the vines in response tostressful conditions. Studies from the Department suggest that thecompound is capable of reducing blood sugar levels by up to 42%, aneffect comparable to that of the antidiabetic drug metformin.Unfortunately for wine drinkers, however, it is unlikely that thecompound is present in wines, due to its instability to light and air.

The potential for cinnamon in treating type 2 diabetes was first notedin August 2000. The news came from an unpublished study demonstratingthat the active compound present in cinnamon, methylhydroxy chalconepolymer (MHCP), could normalise very high glucose levels in diabeticmice. Other experiments suggested this antidiabetic effect of MHCP isdue to its ability to increase the cell's response to insulin, and ithas been estimated that sensitivity to insulin raises by around 20 foldon administration of the chemical. Following the release of theinformation, researchers at the US Agricultural Research Servicerecommended that diabetics consume between one-quarter and one teaspoonof cinnamon per day, either alone or in foods.

Ginsenoside Re, a compound found in the ginseng berry, has been shown tobe beneficial in the treatment of diabetes and obesity, helping tonormalise blood sugar levels, improve insulin sensitivity and aid weightloss. Researchers, from the University of California, tested the effectsof the berry on 2 groups of mice (one nondiabetic set, and a second bredto be diabetic). The berries can help to normalize blood sugar levels,improve insulin sensitivity, lower cholesterol and aid weight loss.Ginsenoside Re has been found to be one of the compounds in the berriesresponsible for the improved insulin sensitivity [BBC News, Monday, 10Apr., 2000; http://news.bbc.co.uk/hi/english/health/newsid2004000/2004255.stm].

Within the scope of the present application the term “insulinsensitizer” or “insulin sensitizing agent” refers to a compound,preferably a pharmaceutical compound or preferably a natural compound,that will lower blood glucose levels by increasing the responsiveness ofthe tissues to preferably insulin.

Insulin sensitizers have been shown to be effective in lowering bloodglucose levels by increasing the responsiveness of target tissues toinsulin. However, as the disease progresses, type 2 diabetic patientsgradually loose their ability to produce sufficient insulin. Thisdecrease in insulin production slowly diminishes the effectiveness ofthe insulin sensitizers, so at a certain point these patients are forcedto change from natural and relatively harmless insulin sensitizers topharmaceutical insulin sensitizers such as biguanides orthiazolidinediones and eventually drastic measures like insulininjections are required to lower blood glucose levels.

To prevent or postpone this need for pharmaceutical insulin sensitizersand finally insulin injections of diabetes type 2, there is a need for acomposition that results in an effective lowering of blood glucose.

SUMMARY OF THE INVENTION

The present invention provides a composition comprising a peptidefraction which is preferably rich in di-and tripeptides in combinationwith an insulin sensitizer preferably a natural or pharmaceuticalinsulin sensitizer. This composition is very suitable as pharmaceuticalor food or a food supplement. Especially this composition is useful forType 2 diabetes. A composition comprising small peptides preferably di-and tripeptides is found to be useful to treat Type 2 diabetes.

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to compositions for the oral treatment ofdiabetes mellitus by lowering the levels of glucose in the bloodcomprising a peptide fraction along with preferably one or more insulinsensitizing agents and to methods for preparing the same. The beneficialeffect of the composition according to the invention is not limited toindividuals suffering from type 2 diabetes but is also recorded forhealthy persons so that it can be applied in the prevention of diabetes.Moreover the composition according to the invention is useful to enhancethe recovery of healthy people after physical exercise. The compositioncan form part of a food, a beverage or a supplement so that it can beused in the manufacture of a large variety of products includingdietetic products, shakes, dietary supplements, infant nutrition,clinical nutrition, beverages such as sports drinks and soft drinks, orother foods, foodstuffs or fermented products.

The fact that the intake of intact protein has a stimulatory role onplasma insulin levels is not new but was already reported in the 1960's.Later work has identified that protein hydrolysates as well as freeamino acids and especially free arginine, leucine, tyrosine andphenylalanine can have strong insulinotropic effects upon theirintravenous injection. Very recently experiments have demonstrated thein-vivo insulinotropic potential of protein hydrolysates in combinationwith free amino acids and carbohydrate upon oral uptake (Van Loon et al,American Journal of Clinical Nutrition, 72:96-105, 2000).

We have observed that the oral intake of intact protein, or proteinhydrolysates, or protein hydrolysates enriched in peptides with amolecular weight below 500 Da, or protein hydrolystes enriched inpeptides with a molecular weight below 500 Da and additionally suppletedwith free amino acids, leads to an increasing insulin response. Of theabovementioned options, intake of carbohydrates in combination withintact protein leads to the lowest insulin response, intake ofcarbohydrates with a protein hydrolysate enriched in in peptides below amolecular weight of 500 Da and additionally suppleted with free aminoacids leads to the highest insulin response.

In line with this observation we have used in our Examples 1 and 4 aprotein source leading to the highest insulin response i.e. a proteinhydrolysate enriched in peptides below a molecular weight of 500 Da andadditionally suppleted with free amino acids to demonstrate thebeneficial effect of a combination with an insulin sensitiser.

At first sight type 2 diabetic patients are expected to benefit from thevigorously stimulated insulin secretion as brought about by the combinedintake of protein hydrolysates, free amino acids, and carbohydrates.Although our own experiments have confirmed this stimulated insulinsecretion, very much to our surprise this enhanced insulin productiondid not result in significant lower blood glucose levels in late stagetype 2 diabetic patients. So the enhanced insulin production alone didnot result in lower glucose levels. Surprisingly our experiments showthat this enhanced insulin production in combination with a suitableinsulin sensitizer does yield the desired lowering of blood glucoselevels. Moreover, the reduction in blood glucose levels as observed withthe composition according to the invention is markedly greater than thelowering effect of the sensitizer alone.

The composition according to the invention with the most potent glucoselevelling effect requires that the insulin sensitizer is suitablycombined with an effective amount of a specific peptide fraction. Toencourage the use of the composition according to the invention in apreventive way, it is highly important that the composition combines ahigh palatability with low costs, i.e. the composition should berelatively cheap and have an acceptable taste.

Optimally suitable peptide fractions are obtained from protein sourceshaving a high content of hydrophobic amino acid residues. Examples ofproteins rich in hydrophobic amino acid residues are milk proteins likewhey or casein, meat proteins, egg proteins, soy proteins, wheatproteins, pea proteins, potato protein, lupine protein, rice proteinsand maize proteins. Preferably the protein raw material is milk protein,soy protein, or maize protein or purified fractions thereof.

In the present context, the term “peptide fraction” is understood toindicate that it may contain all types of peptides that may vary inlength and optionally free amino acids. By “peptides” is meant proteinmolecules having a molecular weight of less than 2000 Da. Thus althoughthe “peptide fraction” may include free amino acids, free amino acidsare not included in the term “peptides”. The preferred peptide fractionis characterized by a peptide size distribution in which the class ofsmall peptides i.e. peptides with a molecular weight below 500 Da, isover-represented. In the peptide fraction according to the inventionthese small peptides are present for at least 30 molar %, preferably forat least 50 molar % and more preferably for at least 70 molar % of allpeptides present with a molecular weight below 2000 Da. Moreover, thepeptide fraction incorporated in the composition according to theinvention is rich in di- and/or tripeptides. Rich in di and/ortripeptides means that at least 20 molar %, preferably at least 30 molar%, more preferably at least 35 molar % of all peptides with a molecularweight below 2000 Da is present as di-and/or tripeptides. The sizedistribution of the peptides composing the peptide fraction can bedetermined using methods as specified in the Materials & Methodssection.

The peptide fraction is preferably obtained by hydrolysing a suitableprotein substrate. The peptide fraction is preferably a proteinhydrolysate containing a high proportion of small peptides, i.e.peptides with a molecular weight below 500 Da. Preferably these peptidefractions are obtained by enzymatic hydrolysis of a plant or animalderived protein or protein fraction.

The protein raw material may be hydrolysed by one or more hydrolyticenzymes. The hydrolytic enzyme can be of animal, plant, yeast, bacterialor fungal origin. Preferably enzyme preparations are used that have abroad cleavage specificity and a low exo-peptidase activity to minimisethe liberation of free amino acids. Preferred enzymes are endoproteases,with such as subtilisin (EC3.4.24.4 or Pescalase as supplied by DSM FoodSpecialities, Seclin, France or Alcalase as supplied by NOVO, Bagsvaerd,Denmark), thermolysin (EC3.4.24.4 or Thermoase as supplied by DaiwaKasei, Osaka, Japan), neutral metallo protease (EC3.4.24.28 or BrewersProtease 2000 as supplied by DSM Food Specialities, Seclin, France orNeutrase as supplied by NOVO) or trypsin (EC 3.4.21.4) or chymotrypsin(EC3.4.21.1). or papain (EC3.4.22.2) or pepsin (EC3.4.23.1). The peptidefraction that can be used to prepare a composition as disclosed in thepresent invention include all protein hydrolysates that can be obtainedby enzymatic hydrolysis or chemical hydrolysis using common techniquesas described in the literature and known to those skilled in the art.

To limit the level of bitter off-tastes that are usually generated uponthe extensive hydrolysis of proteins, the peptide fraction is preferablyobtained by using an amino acid, e.g. a proline-specific, protease. Aproline specific protease implies preferential cleavage at either theaminoterminal or the carboxyterminal side of proline. Endoproteasescapable of cleaving at the aminoterminal side of proline are known(Nature, Vol 391, 15 January 15, pp301-304,1998). Endoproteases with apreference for cleaving at the carboxyterminal side of proline are alsoknown (EC3.4.21.26). The latter type of proline-specific endoprotease ispreferably obtained from food-grade overproducing recombinant strainssuch as Aspergillus. An example of a suitable producer of this enzymehas been described in WO 02/45523. Furthermore proline-specificdipeptidyl-peptidases (EC 3.4.14.2) are known. The use of theseproline-specific proteases in the production of the peptide fractionaccording to the invention has many advantages. Apart from the fact thatoff tastes such as bitterness are prevented, the water solublity of thepeptide fraction is improved and, most importantly, the final peptidesize that can be obtained is considerably reduced. Especially on proteinsubstrates with a relatively high proline content such as wheat gluten(see Example 1) and casein (see Example 4) the incorporation of aproline-specific protease in the hydrolysis process leads to peptidefractions with relatively high proportions of peptides with a molecularweight below 500 Da.

As described in our copending patent application PCT/EP 03/05876 hereinincluded by reference, the use of dipeptidyl- and/ortripeptidyl-peptidases (EC 3.4.14) in the production of hydrolysates isof special importance as these offer an efficient way for producing thepeptide fraction according to the invention. Therefore preferably thepeptide fraction is a hydrolysate preferably comprising a significantamount of di- and/or tripeptides.

As the result of the action of the various proteases used, the peptidefraction is likely to contain free amino acids as well. The amount offree amino acids resulting from the enzymatic hydrolysis reaction mayrange from 1 to 10 wt % of the peptides having a MW of below 2000 Dapresent calculated on dry weight.

Apart from obtaining the peptide fraction by protein hydrolysis, thepeptide fraction can be obtained via chemical or enzymatic synthesis.Moreover a hydrolysate spiked with such synthesised peptides can formthe peptide fraction as present in the composition according to theinvention.

Preferably the peptide fraction is present in the composition accordingto the invention in an amount of 0.5-99 wt %, preferably 1.0-90 wt %,more preferably 1.5-50wt. %, calculated on the basis of the dry weightof the composition.

By ‘insulin sensitizing agent’ or ‘insulin sensitizer’ is meant acompound that will lower blood glucose levels by increasing theresponsiveness of the tissues to insulin. Examples of “natural” insulinsensitizing agents are minerals preferably chromium, vanadium or aB-vitamins like niacin. Furthermore herbs or plant extracts preferablyfrom Banaba leaf, ginseng berry, cinnamon and certain compounds ingrapes have been shown to be effective insulin sensitizers. Also theactive compounds identified in these herbs and plant extracts andthought to be responsible for these insulin sensitising effects arepreferably applied as natural sensitizers: corosolic acid,pterostilbene, methylhydroxy chalcone polymer (MHCP) and GinsensosideRe. Preferred examples of pharmaceutical “insulin sensitizers” arebiguanides (such as Metformin (e.g. Glucophage®) and thiazolidinediones(such as Pioglitazone (e.g. Actos®) and Rosiglitazone (e.g. Avandia®)).These natural insulin sensitizers be preferably added to products inquantities according to their “Reference Daily Intake” or even higher,depending on the person's nutritional need. A list for Reference DailyIntake values is included in the FDA's Code of Federal Regulations 21 CF101.9, Apr. 1, 2001 which includes those nutrients for which a RDI hasbeen established [http://vm.cfsan.fda.gov/-lrd/CFR101-9. HTML].

The composition according the invention may contain a single insulinsensitizing agent or combinations of such agents. Preferably thecomposition according to the invention contains these agents in theirrecommended daily dosages or higher. The composition according to theinvention may contain the peptide fraction and the insulin sensitizer ina mixed form or the peptide fraction and insulin sensitizer may be aseparately packed and sold as a one package. Also the use of thecomposition according to the invention in combination with apharmaceutical insulin sensitiser may be recommended.

Apart from peptides and the insulin sensitizing agent, the compositionmay optionally contain free amino acids. Free amino acids belonging tothe group of leucine and/or arginine and/or phenylalanine and/ortyrosine are of particular importance. These free amino acids may beadded to the peptide fraction to obtain the composition according to theinvention. Because in some countries the addition of extra free aminoacids is not allowed, the desired balance between the bound and freeamino acids can also be obtained using selective combinations of anendoprotease plus an exoprotease as outlined in WO02/32232. In this casethe free amino acids present have been obtained by a further proteolyticbreakdown of the peptide fraction and form an integral part of thepeptide fraction. Preferred compositions are rich in amino acids fromthe group of leucine and/or arginine and/or phenylalanine and/ortyrosine. Preferably the total of the free and peptide bound amino acidsbelonging to the group of leucine, arginine, phenylalanine and tyrosineare present in the composition of the invention in at least 10% wt,preferably at least 20% wt, more preferably at least 30% wt, still morepreferably at least 50 wt % and most preferably at least 60 wt %,calculated on the basis of the dry weight of the total of free andpeptide bound amino acids. Preferably the amino acids from this groupare present in the composition in an amount of 0.5-99 wt %, preferably1.0-90 wt %, more preferably 1.5-50 wt %, calculated on the basis of thedry weight basis of the composition.

The level of free amino acids as well as the total content of aminoacids present in the final composition can be established using methodsspecified in the Materials & Methods section.

Apart from amino acids, carbohydrates are optionally present in thecomposition according to the invention. Depending upon the anticipateduse, i.e. as such or in combination with other food, the composition maycontain a separate source of carbohydrates. These carbohydrates can beglucose or more slowly absorbed carbohydrates like maltodextrins orstarch depending upon the desired glycaemic- index for the particularapplication. In the composition of the invention, carbohydrates can bepresent in an amount of 1.0-90% wt, preferably 2-50% wt, more preferably6-35% wt calculated on the basis of dry weight of the composition.

The wide range of carbohydrate content of the composition according tothe invention can be explained by different anticipated uses of thecomposition.

On the one hand, an optimized formulation for consumers that prefer totake the composition alongside their carbohydrate containing meal oreven prefer to take the composition in between meals is an almost purepeptide and insulin sensitizer supplement, e.g. in the form of a tablet,a syrup or a sachet.

On the other hand, the composition may be formulated so that it can beintegrated in a carbohydrate containing regular meal or “approved meal”.

Other optional components of the composition according to the inventionare vitamins, minerals, flavours, antioxidants, components havingco-enzyme and antioxidant properties, lipids including emulsifiers,colorants, and proteins for meeting specific nutritional and/orphysiological needs.

The composition of the present invention can be either a pharmaceuticalcomposition or a food composition.

The composition according to the invention may be a solid of liquid. Thecomposition may have the form of a powder, a tablet, a capsule, othergalenic forms, a beverage or any other food product.

The composition of the present invention may be part of a normal meal orpart of an approved meal.

By “approved meal” or “approved diabetic diet” is meant a mealrecommended or approved by a national nutritional organization for thehealth of a diabetic, for example, the American Diabetes AssociationInc. (“ADA”). For example the meals recommended in “Maximizing the Roleof Nutrition in Diabetes Management” published 1994 by the AmericanDiabetes Association, Inc., are “approved meals”. Other Westernorganizations, which recommend or approve a meal for a diabetic, are TheInternational Diabetes Federation; the European Association For theStudy of Diabetes; and the European and Canadian Dietetic Association.Other Eastern and Far Eastern organizations are the Chinese DiabetesFederation; the Japanese Diabetes Federation; and the Indian DiabetesFederation. It will be appreciated that an “approved meal” will varydepending upon the culture and geography of the diabetic. However, it isunderstood that, irrespective of either culture of geography, acompliant diabetic will eat a meal which makes no more than a reasonabledemand upon his/her system.

LEGENDS TO THE FIGURES

FIG. 1 shows the Plasma insulin response (microUnits/ml) for type 2diabetic patients in minutes after drinking a beverage containingcarbohydrate (CHO) or carbohydrate plus peptides and free amino acids(CHO+PRO) respectively.

FIG. 2 shows the Plasma glucose response (mmol/l) for type 2 diabeticpatients in minutes after drinking a beverage containing carbohydrate(CHO) or carbohydrate plus peptides and free amino acids (CHO+PRO)respectively.

FIG. 3 shows the Plasma insulin response (microUnits/ml) for type 2diabetic patients in minutes after drinking a beverage containingcarbohydrate (CHO+insulin sensitiser) or carbohydrate plus peptides andfree amino acids (CHO+PRO+insulin sensitiser) while taking an insulinsensitizer simultaneously.

FIG. 4 shows the Plasma glucose response (mmol/l) for type 2 diabeticpatients in minutes after drinking a beverage containing carbohydrate(CHO+insulin sensitiser) or carbohydrate plus peptides and free aminoacids (CHO+PRO+insulin sensitiser) while taking an insulin sensitizersimultaneously.

FIG. 5 shows LC/MS/MS data of a lactalbumin hydrolysate, obtained usingDelvolase

FIG. 6 shows LC/MS/MS data of a lactalbumin hydrolysate, obtained usingDelvolase+TPAP-A

FIG. 7 shows LC/MS/MS data of a lactalbumin hydrolysate, obtained usingDelvolase+proline specific endoprotease (EndoPro)

FIG. 8 shows LC/MS/MS data of a lactalbumin hydrolysate, obtained usingDelvolase+TPAP-A+proline specific endoprotease (EndoPro).

EXAMPLES

Materials and Methods

Sodium caseinate containing 90% protein was obtained from DMVInternational (The Netherlands). Wheat gluten was obtained from Cargill.L-Leucine, L-Phenylalanine, beta-lactoglobulin and crude lactalbuminwere obtained from Sigma-Aldrich.

Subtilisin from B.licheniformis (Delvolase®), 560 000 DU per gram) wasobtained from DSM Food Specialities (Seclin, France).

Enzyme Activity

Overproduction and chromatographic purification of the proline specificendoprotease from Aspergillus niger was accomplished as described in WO02/45524. The activity of the latter enzyme was measured according tothe method described in Japanese patent JP5015314 with minormodifications. Briefly, the enzymatic activity was tested onZ-Gly-Pro-pNA at 37 degrees C. in a citrate/disodium phosphate buffer pH5. pH 5.0 was chosen because the pH optimum of the enzyme is below pH 6in this test. The reaction product was also monitoredspectrophotometrically at 410 nM. Overproduction and chromatographicpurification of the tripeptidylpeptidase TPAP-A encoded by gene 12 fromAspergillus niger was accomplished as described in EP 02100667.1. Theactivity of the purified tripeptidyl aminopeptidase was measured in asimilar way. However, in this case the synthetic substrateAla-Ala-Phe-pNA (Bachem, Switserland) was used in an incubation in 0.1mol/litre citrate buffer at pH 4.0 and 60 degrees C. The purified TPAP-Ahad an activity of 8 units/ml.

A unit is defined as the quantity of enzyme that provokes the release of1 μmol of p-nitroanilide per minute under these conditions.

The Degree of Hydrolysis (DH) as obtained during incubation with thevarious proteolytic mixtures was monitored using a rapid OPA test (JFS,Vol 66, NO 5, 2001).

The average peptide chain length in the various peptide fractions wasdetermined by chromatography over a Superdex Peptide HR 1030 column.

Sensoric Evaluation of Peptide Fraction “Bitterness”

Sensoric evaluation of the various peptide fractions was carried out byan independent panel trained in detecting and ranking various levels ofbitterness. During the sessions, the taste trials were performed ‘blind’and bitterness was scored on a scale from 0 (none)—4 (very bitter).Panel members were trained with quinine sulphate with the followingsolutions:

15 ppm quinine sulphate>Intensity bitter=1

20 ppm quinine sulphate>Intensity bitter=2

30 ppm quinine sulphate>Intensity bitter=3

50 ppm quinine sulphate>Intensity bitter=4

Peptide size distribution of peptide fractions

The content of peptides with a molecular weight below 500 kDa isestimated using a HPSEC method using a Superdex HR10/30 AmershamPharmacia (range 100-7000 Da) in combination with the required referencemolecules. The content of di- and tripeptides is estimated using thevolatile ion-pairing reagent NFPA in combination with reversed phaseliquid chromatography as described in the LC/MS analysis section.

LC/MS Analysis.

HPLC using an ion trap mass spectrometer (Thermoquest®, Breda, theNetherlands) coupled to a P4000 pump (Thermoquest®, Breda, theNetherlands) was used in characterising the enzymatic proteinhydrolysates produced by the inventive enzyme mixture. The peptides wereseparated using a PEPMAP C18 300A (MIC-15-03-C18-PM, LC Packings,Amsterdam, The Netherlands) column in combination with a gradient of0.1% formic acid and 1 mM nonafluoropentaoic acid (NFPA) in Milli Qwater (Millipore, Bedford, Mass., USA; Solution A) and 0.1% formic acidin acetonitrile (Solution B) for elution. The gradient started at 95% ofSolution A and increased to 40% of solution B in 140 minutes and waskept at the latter ratio for another 5 minutes. The injection volumeused was 50 microliters, the flow rate was 50 microliter per minute andthe column temperature was maintained at 30° C. The proteinconcentration of the injected sample was approximately 50micrograms/milliliter.

Detailed information on the individual peptides was obtained by usingthe “scan dependent” MS/MS algorithm, which is a characteristicalgorithm for an ion trap mass spectrometer.

Full scan analysis was followed by zoom scan analysis for thedetermination of the charge state of the most intense ion in the fullscan mass range. Subsequent MS/MS analysis of the latter ion resulted inpartial peptide sequence information, which could be used for databasesearching using the SEQUEST application from Xcalibur Bioworks(Thermoquest®, Breda, The Netherlands). Databanks used were extractedfrom the OWL.fasta databank, available at the NCBI (National Centre forBiotechnology informatics), containing the proteins of interest for theapplication used. In those experiments in which well characterizedprotein substrates such as whey proteins or caseins were measured, theprecision of the analysis technique was increased by omitting thoseMS/MS spectra with a sequence fit of less than 50%.

By using different enzyme mixtures the mass range of the peptides formedstarted at di- and tripeptides. By using the volatile ion-pairingreagent NFPA in combination with reversed phase liquid chromatographysmaller and more hydrophilic peptides with a mass ranging from approx.200 to 2000 Daltons can be monitored. This was considered suitable forfurther analysis by MS sequencing.

Angiotensin (M=1295.6) was used to tune for optimal sensitivity in MSmode and for optimal fragmentation in MS/MS mode, performing constantinfusion of 60 mg/ml, resulting in mainly doubly and triply chargedspecies in MS mode, and an optimal collision energy of about 35% inMS/MS mode.

Determination of Amino Acids

A precisely weighed sample of the proteinaceous material was dissolvedin dilute acid and precipitates were removed by centrifugation in anEppendorf centrifuge. Amino acid analysis was carried out on the clearsupernatant according to the PicoTag method as specified in theoperators manual of the Amino Acid Analysis System of Waters (MilfordMass., USA). To that end a suitable sample was obtained from the liquid,added to dilute acid and homogenized. From the latter solution a newsample was taken, dried and derivatised using phenylisothiocyanate. Thevarious derivatised amino acids present were quantitated using HPLCmethods and added up to calculate the total level of free amino acids inthe weighed sample.

To relate this total level of free amino acids in the sample to thetotal level of amino acids that can be liberated from this sample, thesample is also subjected to acid hydrolysis followed by a quantificationof the total free amino acids present as detailed above. Since duringacid hydrolysis Trp and Cys are destroyed, these amino acids are notincluded in the data presented. However, Gln and Asn residues areconverted into Glu and Asp during acid hydrolysis so that the values forGlu and GIn, and for Asp and Asn were usually summed together to allowcomparison. with the data obtained before acid hydrolysis.

Example 1 Peptides Enriched with Di- and Tripeptides and Amino AcidsEnhance Plasma Insulin Levels but do not Diminish High Glucose Levels inDiabetic Patients

The aim of this study was to investigate if a peptide fraction enrichedwith free leucine and phenylalanine could significantly lower bloodglucose levels in type 2 diabetic patients.

Subjects

Ten (long-term) diagnosed male type 2 diabetic patients were selected toparticipate in this study. Exclusion criteria were impaired renal orliver function, obesity (BMI>30), cardiac disease, hypertension,diabetic complications and (exogenous) insulin therapy. Most subjects(n=8) were using oral antidiabetics. In the diabetic subjects,antidiabetic medication was withheld for 3 days before participation inthe trials and throughout the entire experimental period. Subjects werescreened for glucose intolerance/type 2 diabetes by an oral glucosetolerance test (OGTT) according to the World Health Organisationcriteria of 1999.

Study Design

Each subject participated in 2 trials, separated by one week, in whichthe insulin response and the glucose response to the ingestion of 2drink compositions (CHO or CHO+PRO; see below for definitions) wasdetermined. Both trials lasted 2 hours in which subjects were seated andremained inactive. Drinks were provided in randomised order and doubleblind.

Protocol

Experiments were carried out at 8:30 A.M after an overnight fast. ATeflon catheter was inserted into an antecubital vein and a restingblood sample was drawn (t=0 minutes). Immediately thereafter subjectsdrank an initial bolus (3 ml.kg⁻¹) of a given test drink (CHO or CHO+PROtrial). Repeated boluses (3 ml.kg⁻¹) were taken every 30 minutes untilt=90 minutes. Blood samples were drawn at 15 minutes intervals formeasurement of plasma glucose and insulin concentrations.

Beverages

At t=0, t=30, t=60 and t=90 subjects received a beverage volume of 3ml/kg body weight to ensure a given dose of 0.8 g/kg body weight/hourcarbohydrate (50% glucose; 50% maltodextrin) without or with anadditional 0.4 g/kg body weight/hour of a peptide fraction (CHO orCHO+PRO trial, respectively). This peptide fraction consisted of anextensively hydrolysed wheat protein (0.2 g/kg body weight/hour)suppleted with free leucine (0.1 g/kg body weight/hour) as well as freephenylalanine (0.1 g/kg body weight/hour). So half of the nitrogen assupplied by the beverage was supplied by wheat protein peptides, onequarter by the free leucine and another quarter by the freephenylalanine.

Analysis

Blood (10 ml) was collected in EDTA containing tubes and centrifuged at1000 g and 4° C. for 10 minutes. Aliquots of plasma were frozenimmediately in liquid nitrogen and stored at −80° C. Glucose (Unit KitIII, 07367204, Roche, Basel, Switzerland) was analysed with the COBASFARA semi automatic analyser (Roche, Basel, Switzerland). Insulin wasanalysed by radio-immuno-assay (Insulin RIA 100 kit, Pharmacia, Sweden).

According to the results obtained (see FIG. 1) the insulin responses ofthe type 2 diabetic patients were substantially increased by theinclusion of the peptide fraction (PRO) mixture in the drink. Theinsulin levels increased to 289% of the original 100% (P<0.01). Theresults showed that while keeping the glucose intake the same, additionof the peptide fraction results in a normalisation of the insulinresponse in type 2 diabetics (see FIG. 1). However, unlike the insulinresponse, plasma glucose concentrations were not differently affectedbetween trials within this timeframe (see FIG. 2) These observationslead to the surprising observation that although the insulin response inlong-term diagnosed type 2 diabetic patients was substantially enhancedby the addition of the peptides plus free amino acids, no significantlowering effect was observed on their blood glucose concentrations.

Example 2 Beta-Casein Hydrolysates Obtained by a Proline-SpecificEndoprotease in Combination with a Tripeptidyl Aminopeptidase ContainHigh Proportions of Tripeptides as well as Peptides havingCarboxyterminal Proline Residues

To allow a more precise LC/MS/MS analysis of the various reactionproducts obtained by combining a proline-specific endoprotease with atripeptidylpeptidase on proline rich protein substrates such as wheatgluten or casein, another hydrolysis experiment was carried out in whichpure bovine beta casein was used as the substrate. To that end a 0.2%(w/w on protein) solution was prepared by dissolving pure beta-casein(Sigma) in water and adjusting the pH to 8.0 by NaOH. Then the serineprotease subtilisin (Delvolase) was added to a concentration of 5%(volume of the commercial enzyme product per weight of beta-casein) andthe mixture was incubated for 1 hour at 60 degrees C. under non-pH-statconditions. The reaction was stopped by lowering the pH to 5.5 usinglactic acid followed by a heat treatment of 10 minutes at 90 degrees C.Then the mixture was cooled down to 50 degrees C. and a sample was takenfor LC/MS/MS analysis. A subsequent incubation with the proline-specificendoprotease (EndoPro) from A. niger (see WO 02/45523) was carried outby adding a chromatographically purified solution of the overproducedproline specific endoprotease from A. niger in a concentration of 20units/gram protein. After incubating for 2 hours at 50 degrees C. undernon-pH-stat conditions the proline specific endoprotease was inactivatedby another heat treatment to yield another sample for LC/MS/MS analysis.Finally chromatographically purified TPAP-A (see EP 02100667.1) wasadded in a concentration of 4 units per gram substrate and theincubation was continued for 2 hours at 60 degrees C. and theninactivated by heating to yield another LC/MS/MS sample. Subsequentincubations were carried out on beta-casein without Delvolase using theproline-specific endoprotease and TPAP-A, either alone or incombination, under the above described conditions. The latter sampleswere also subjected to LC/MS/MS analysis. The data obtained are shown inthe Table underneath. Peptides with C- Enzymes used Number terminal-ProDi + tripeptides Tripeptides to prepare of (molar % of all (molar % ofall (molar % of all beta-casein peptides peptides peptides peptideshydrolysate analysed analysed) analysed) analysed) Subtilisin 93 0 12 6Subtilisin + 68 41 34 25 EndoPro Subtilisin + 69 36 45 36 EndoPro +TPAP-A EndoPro 55 49 11 11 TPAP-A 1 0 100 100 EndoPro + TPAP- 68 40 4340 ADespite the broad specificity of the TPAP-A enzyme used (see EP02100667.1), incubating the pure beta-casein with just the TPAP-A enzymeresults in the release of a single peptide only, i.e. the N-terminaltripeptide Arg-Glu-Leu of beta-casein. For an unknown reason the TPAP-Aenzyme used cannot remove the next tripeptide from this substrate herebyclearly demonstrating the need for combining the TPAP with anendoprotease such as subtilisin or a proline specific endoprotease or acombination of the two. The results shown above clearly indicate thatsuch combinations of a tripeptidylpeptidase with one or moreendoproteases lead to a considerable increase in the number oftripeptides generated. Combinations involving proline specificendoproteases all show an impressive increase in the number of peptideshaving carboxyterminal proline residues from which a reduced bitternessmay be inferred (see WO 02/45523).

Example 3 Benefits of Combining a Proline-Specific Endoprotease with aTripeptidylpeptidase on Substrates Low in Proline

Here we demonstrate that the enzyme combination of a proline-specificendoprotease in combination with a tripeptidyl peptidase is alsobeneficially used in the hydrolysis of substrates with lower prolinecontents.

A crude lactalbumin fraction from bovine milk (Sigma) was suspended inwater in a concentration of 20 grams/liter after which the pH wasadjusted to 8.0. The serine protease subtilisin (Delvolase) was added toa concentration of 4% (volume of the commercial enzyme product perweight of the substrate) and the mixture was incubated for 2 hours at 60degrees C. under non-pH-stat conditions. Then the pH of the suspensionwas lowered to pH to 4.5 using citric acid and divided into 4 portions.One portion was heated to inactivate the Delvolase enzyme and then keptfrozen until LC/MS/MS analysis. To the other three portions either thechromatographically purified proline specific endoprotease from A.niger(EndoPro) was added (1 unit/gram lactalbumin) or tripeptidylpeptidase(TPAP-A; 20 units/gram of lactalbumin) or a combination of prolinespecific endoprotease and TPAP-A (1 unit+20 units/gram lactalbumin; seeMaterials&Methods for unit definitions). The mixtures were incubatedovernight at 50 degrees C., subjected to a heat treatment to inactivatethe enzymes and stored at −20 degrees C. Samples were first centrifugedand the clear supernatant was used for LC/MS/MS analysis. Only thosepeptides fitting with the amino acid sequence of alpha-lactabumin weretaken into account.

The LC/MS/MS data obtained are shown in FIGS. 5, 6, 7 and 8: peptidelength in amino acid residues is depicted on the X-axis and on theY-axis the number of peptides analysed. Even without a recalculation ofpeptides of a specific length into percentages of the total peptidesanalysed, the benefits of an incubation with either an proline-specificendopeptidase or a tripeptidyl peptidase or a combination of these twoenzymes, become visible. Together with the results provided in theprevious Example the data obtained clearly demonstrate that thecombination of a proline-specific endoprotease with a tripeptidylpeptidase provides hydrolysates enriched in peptides with a molecularweight below 500 Da and, more specifically, enriched in di- andtripeptides, be it on proline-rich or on other proteinaceous substrates

Example 4 Peptides Enriched with Di- and Tripeptides Combined with anInsulin Sensitizer Raise Plasma Insulin Levels and Diminish High GlucoseLevels in Diabetic Patients

The aim of this study is to investigate if a peptide fraction accordingto the invention in combination with an ‘insulin sensitizer’ can raisethe insulin response in type 2 diabetic patients, thereby lowering bloodglucose levels. The peptide fraction used was obtained by extensivelyhydrolysing sodium caseinate to obtain a peptide fraction containingmore than 70 molar % of peptides with a molecular weight below 500 Dasuppleted with levels of free leucine and free phenylalanine identicalto the levels used in Example 1.

Subiects

Six (long-term) diagnosed male type 2 diabetic patients all using oral‘insulin sensitizer’ drugs (but no exogenous insulin) are selected toparticpate in this study. Exclusion criteria are impaired renal or liverfunction, obesity (BMI>30), cardiac disease, hypertension, diabeticcomplications and (exogenous) insulin therapy.

Each subject participates in 2 trials, seperated by one week, in whichthe insulin and glucose response to the ingestion of 2 beveragecompositions (Carbohydrate (CHO) or Carbohydrate+Peptide fractionaccording to the invention (CHO+PRO)) is determined while the diabeticpatients maintain their usual ‘insulin sensitizer’ therapy. Both trialslast 3 hours in which subjects are seated and are remained inactive.Drinks are provided in randomised order and double blind.

Protocol

Experiments are carried out at 8:30 A.M. after an overnight fast. ATeflon catheter is inserted into an antecubital vein and a resting bloodsample is drawn (t=0 minutes). Immediately thereafter subjects drink aninitial bolus (3 ml.kg⁻¹) of a given test drink (CHO or CHO+PRO trial).Repeated bolusses (3 mi.kg⁻¹) are taken every 30 minutes. Blood samplesare drawn at 15 minutes intervals for measurement of plasma glucose andinsulin concentrations.

Beverages

Beverage volumes and dosages of carbohydrate (CHO) and carbohydrate pluspeptides and free amino acids (CHO+PRO) were as described in Example 1.

Analysis

Blood samples (10 ml) are collected in EDTA containing tubes andcentrifuged at 1000 g and 4 degree Celsius for 10 minutes. Aliquots ofplasma are frozen immediately in liquid nitrogen and stored at −80degree Celsius. Glucose (Unit Kit III, 07367204, Roche, Basel,Switzerland) is analysed with the COBAS FARA semi automatic analyser(Roche, Basel, Switzerland). Insulin is analysed by radio-immuno-assay(Insulin RIA 100 kit, Pharmacia, Sweden).

In contrast with the results recorded in Example 1, the combined use ofan insulin sensitizer and an amino acid-protein hydrolysate compositionaccording to the invention results in an enhanced plasma insulinresponse (see FIG. 3) plus a significantly lowered blood glucose levelin type 2 diabetic patients (see FIG. 4).

1. A composition comprising an insulin sensitizer and a peptidefraction.
 2. A composition according to claim 1, wherein the peptidefraction is part of a hydrolysate.
 3. A composition according to claim1, wherein the peptide fraction comprises an amount of at least one freeamino acid which is any of leucine, phenylalanine and arginine.
 4. Acomposition according to claim 1 wherein the peptides of the peptidefraction are protein molecules having a molecular weight of less than2000 Da.
 5. A composition according to claim 1 wherein the peptidefraction is rich in peptides having a molecular weight below 500 Dapreferably rich in di and/or tripeptides.
 6. A composition according toclaim 5 wherein at least 20 molar %, at least 25 molar % or at least 30molar % or at least 35 molar % of the peptides with a molecular weightbelow 2000 Da, is present as di and/or tripeptide.
 7. A compositionaccording to claim 5, wherein the di-and/or tripeptides are rich inproline at one end of the peptide.
 8. A composition according to claim5, wherein at least 20%, at least 30% or at least 40% of the prolinepresent in the starting protein is present in the di and/or tripeptides.9. A composition according to claim 5, wherein 30% of the tripeptides or35% of the tripeptides have a carboxy terminal proline.
 10. Acomposition according to claim 5, wherein at least 30 molar % of thepeptides or at least 50 molar % or at least 70 molar % of all peptidespresent with a molecular weight below 2000 Da.
 11. A compositionaccording to claim 1, wherein the insulin sensitizer is chromium,vanadium, niacin, corosilic acid, banana leafextract, ginseng berry,Ginsensoside Re, cinnamon, methylhydroxy chalcone polymer,pterostilbene, biguanide or thiazolidinedione.
 12. A dietic product, apharmaceutical product, or a food or a food supplement comprising thecomposition according to claim
 1. 13. A composition according to claim 1for use in a method of treatment of the human or animal body by therapyor diagnosis.
 14. Use of a composition according to claim 1 for themanufacture of a medicament for treating type 2 diabetes or preventingdiabetes.
 15. Use of a composition comprising a peptide fraction for themanufacture of a medicament for treating type 2 diabetes or preventingdiabetes.
 16. Use according to claim 15, wherein the peptide fractioncomprises at least one free amino acid which is one of tyrosine,leucine, phenylamine and arginine.
 17. Method of treatment of type 2diabetes which comprises the use of a composition according claim
 1. 18.Method of preventing diabetes which comprises the use of a compositionaccording to claim 1